Biology subjects

Holzbaur, E. L. F.

Publications and source records attributed to Holzbaur, E. L. F..

14 recordsLinked to original sources

bioRxiv2026

PINK1/Parkin-dependent mitophagy mediates astrocytic inflammatory responses to mitochondrial damage

Astrocytes directly influence neuronal survival and increasingly are understood to contribute to the progression of neurodegenerative diseases including Parkinsons disease (PD). Mitochondrial damage is a hallmark of PD pathology in both neurons and astrocytes. Damaged mitochondria are cleared by PINK1/Parkin-mediated mitophagy; loss-of-function mutations in either PINK1 or Parkin are sufficient to cause PD. Neuronal mitophagy is well-studied, but far less is known about how mitochondrial dysfunction in astrocytes affects neural health. While microglial release of pro-inflammatory cytokines has been shown to induce astrocytes to mount their own inflammatory response, we hypothesize that a more direct pathway is involved, and that mitochondrial damage to astrocytes directly triggers release of proinflammatory cytokines. To address these questions, we treated primary murine cortical astrocytes with oxidative phosphorylation (OXPHOS) inhibitors antimycin A (AA) and oligomycin A (OA) and observed the PINK1-dependent accumulation of Parkin on damaged mitochondria, leading to phospho-ubiquitination of proteins in the outer mitochondrial membrane and the recruitment of the autophagy receptor SQSTM1/p62. To identify transcriptional changes caused by mitochondrial damage and the resulting activation of mitophagic machinery, we performed bulk RNA-sequencing on astrocytes isolated from WT, PINK1-/-, or Parkin-/- mice treated with AA/OA or a vehicle control. In WT astrocytes, TNF- signaling via NF-{kappa}B was the most significantly upregulated pathway following OXPHOS inhibition. OXPHOS inhibitor treatment also stimulated p62 expression, while NF-{kappa}B inhibition prevented this upregulation. Astrocytic secretion of cytokines, including TNF-, was increased following mitochondrial damage; this secretion was dependent on NF-{kappa}B activation and occurred at levels sufficient to induce mitochondrial depolarization in hippocampal neurons. Compared to WT astrocytes, PINK1-/- astrocytes showed a significant reduction in transcriptional signatures associated with TNF- signaling following mitochondrial damage, while Parkin-/- astrocytes exhibited upregulation of both IFN-{gamma} and IFN- signaling. These findings indicate altered inflammatory responses to mitochondrial damage in the absence of functional PINK1 or Parkin. Finally, we analyzed scRNA-sequencing data from substantia nigra astrocytes harvested from human brain tissue from PD-positive or control samples. Distinct clusters comprised predominantly of PD-positive or control astrocytes emerged. Astrocytes in the PD-positive cluster were enriched for NF-{kappa}B, IFN- and IFN-{gamma} responses, consistent with the signaling observed in vitro post-OXPHOS inhibition. Together, these findings identify inflammatory signatures activated by mitochondrial damage in astrocytes, and establish this pathway as a potential contributor to neuroinflammation in PD.

cell biology↗

bioRxiv2024

Mitochondrial damage triggers concerted degradation of negative regulators of neuronal autophagy

Mutations in genes that regulate mitophagy, a key mitochondrial quality control pathway, are causative for neurological disorders including Parkinsons. Here, we identify a novel stress response pathway activated by mitochondrial damage that regulates mitophagy in neurons. We find that increasing levels of mitochondrial stress triggers a graded, concerted response that induces proteasomal degradation of negative regulators of autophagy. These include Myotubularin-related phosphatase 5 (MTMR5), MTMR2 and Rubicon. This Mitophagic Stress Response (MitoSR) pathway is neuron-specific and acts in parallel to the classical Pink1/Parkin-mediated mitophagy pathway. While MTMR5/MTMR2 inhibits autophagosome biogenesis, we find that Rubicon inhibits lysosomal function and thus blocks autophagosome maturation. Targeted depletion of these negative regulators is sufficient to enhance mitophagy, promoting autophagosome biogenesis and facilitating the fusion of mitophagosomes with lysosomes. Our work suggests that therapeutic activation of the MitoSR pathway to induce degradation of negative regulators of autophagy may enhance mitochondrial quality control in stressed neurons.

neuroscience↗

bioRxiv2023

Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery

Neurons rely on long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, and how efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule severing enzyme spastin in locally enhancing microtubule polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components, termed protosynapses, which are distributed along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new microtubule growth and synaptic vesicle precursor (SVP) pausing/retention. Disruption of neuronal spastin, either by CRISPRi-mediated depletion or transient overexpression, interrupts the localized enrichment of dynamic microtubule plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of microtubule polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.

cell biology↗

bioRxiv2023

RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors

Gain-of-function mutations in the LRRK2 gene cause Parkinsons disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adapter MADD, potentially preventing formation of the RAB3A-MADD-KIF1A/1B{beta} complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD. SUMMARYDou et al. demonstrate that Parkinsons disease-associated hyperactive LRRK2 decreases trafficking of synaptic vesicle proteins within neurons by disrupting regulation of the synaptic vesicle precursor protein RAB3A. Impaired delivery of synaptic proteins to presynaptic sites could contribute to progression of debilitating non-motor PD symptoms.

cell biology↗

bioRxiv2023

Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases

Neuronal autophagosomes, "self-eating" degradative organelles, form at presynaptic sites in the distal axon and are transported to the soma to recycle their cargo. During transit, autophagic vacuoles (AVs) mature through fusion with lysosomes to acquire the enzymes necessary to breakdown their cargo. AV transport is driven primarily by the microtubule motor cytoplasmic dynein in concert with dynactin and a series of activating adaptors that change depending on organelle maturation state. The transport of mature AVs is regulated by the scaffolding proteins JIP3 and JIP4, both of which activate dynein motility in vitro. AV transport is also regulated by ARF6 in a GTP-dependent fashion. While GTP-bound ARF6 promotes the formation of the JIP3/4-dynein-dynactin complex, RAB10 competes with the activity of this complex by increasing kinesin recruitment to axonal AVs and lysosomes. These interactions highlight the complex coordination of motors regulating organelle transport in neurons. SummaryMature autophagosomes in the axon are transported by the microtubule motor dynein, activated by JNK-interacting proteins 3 and 4 (JIP3/4). This motility is regulated by the small GTPases ARF6 and RAB10. The tight regulation of autolysosomal transport is essential for intracellular recycling to maintain neuronal homeostasis.

cell biology↗

bioRxiv2022

Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes

Gain-of-function mutations in the LRRK2 gene cause Parkinsons disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We found that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin motors. In iPSC-derived human neurons, knock-in of the strongly-hyperactive LRRK2-p.R1441H mutation caused striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knock-out of the opposing Protein Phosphatase 1H (PPM1H) phenocopied the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuated transport defects in both p.R1441H knock-in and PPM1H knock-out neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive "tug-of-war" between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy.

neuroscience↗

bioRxiv2022

The selective autophagy receptor p62 and the heat shock protein HSP27 facilitate lysophagy via the formation of phase-separated condensates

Lysosomes are membrane-bound organelles that regulate cellular proteostasis. Loss of lysosomal integrity initiates cell death pathways. Thus, cells must rely on quality control mechanisms for protection, including the selective isolation and degradation of damaged lysosomes by lysophagy. Here, we report that the selective autophagy receptor SQSTM1/p62 is an essential lysophagy receptor recruited to damaged lysosomes in both HeLa cells and neurons. p62 oligomers form liquid-like condensates that are critical in lysophagy. These condensates are regulated by the small heat shock protein HSP27, which binds p62 to prevent p62 aggregation and facilitate autophagosome formation. Mutations in p62 are implicated in Amyotrophic Lateral Sclerosis (ALS), and expression of ALS-associated mutations in p62 impair lysophagy, suggesting that deficits in this pathway may contribute to the cellular pathogenesis of ALS. Thus, p62 oligomers cooperate with HSP27 to promote lysophagy by forming a platform for autophagosome biogenesis at damaged lysosomes.

cell biology↗

bioRxiv2022

Damaged mitochondria recruit the effector NEMO to activate NF-κB signaling

Failure to clear damaged mitochondria via mitophagy disrupts physiological function and may initiate damage signaling via inflammatory cascades. However, signaling mechanisms leading from impaired mitophagy to neuro-inflammation are unclear. We discovered that NF-{kappa}B essential regulator NEMO is recruited to damaged mitochondria in a Parkin- and p62/SQSTM1-dependent manner in a time-course similar to recruitment of the structurally-related mitophagy receptor, OPTN. NEMO and p62 colocalize, partitioning into distinct domains from OPTN. Either depletion of p62 or mutation of NEMOs ubiquitin-binding domain abolishes NEMO recruitment, indicating multifactorial interactions. The active catalytic IKK component phospho-IKK{beta} colocalizes with NEMO on damaged mitochondria, initiating NF-{kappa}B signaling and the upregulation of inflammatory cytokines. These findings suggest that damaged mitochondria serve as an intracellular platform for innate immune signaling by promoting the formation of activated IKK complexes in a Parkin-dependent manner. We propose that mitophagy and NF-{kappa}B signaling are competing pathways regulating the response to cellular stress.

cell biology↗

bioRxiv2022

Spatiotemporal analysis of axonal autophagosome-lysosome dynamics reveals limited fusion events trigger two-step maturation

Macroautophagy is a homeostatic process required to clear cellular waste including aggregated proteins and dysfunctional organelles. Neuronal autophagosomes form constitutively in the distal tip of the axon and are actively transported toward the soma, with cargo degradation initiated en route. Cargo turnover requires autophagosomes to fuse with lysosomes to acquire degradative enzymes; however, the timing and number of these fusion events in the axon have proven difficult to detect using microscopy alone. Here we use a quantitative model, parameterized and validated using data from live and fixed imaging of primary hippocampal neurons, to explore the autophagosome maturation process on a cellular scale. We demonstrate that retrograde autophagosome motility is independent from lysosomal fusion, and that most autophagosomes fuse with only a few lysosomes by the time they reach the soma. Furthermore, our imaging and model results highlight the two-step maturation of the autophagosome: fusion with a lysosome or late endosome is followed by the slow degradation of the autophagosomal inner membrane before actual cargo degradation can occur. Together, rigorous quantitative measurements and mathematical modeling elucidate the dynamics of autophagosome-lysosome interaction and autophagosomal maturation in the axon.

cell biology↗

bioRxiv2021

Structural Basis for Membrane Recruitment of ATG16L1 by WIPI2 in Autophagy

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed {beta}-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 [A] resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 {beta}-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.

cell biology↗

bioRxiv2021

ALS and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy

TANK-binding kinase 1 (TBK1) is a multi-functional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD), including missense mutations that disrupt the ability of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), auto-activate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presense of either WT- or kinase inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals. SIGNIFICANCE STATEMENTMissense mutations in TANK-binding kinase 1 (TBK1) have various biophysical and biochemical effects on the molecule, and are associated with the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). TBK1 plays an essential role in clearing damaged mitochondria. Here, we investigate the impact of 10 ALS-linked TBK1 mutations on the critical early stage of mitophagy. We find that both TBK1 recruitment and kinase activity contribute to the clearance of the damaged mitochondria. Furthermore, in neurons, expression of TBK1 mutants alone affects mitochondrial network health. Our investigation utilizes disease-linked mutations to further refine the current model of mitophagy, identifying crosstalk between the regulatory kinases TBK1 and ULK1, and providing new insights into the roles of TBK1 in neurodegenerative pathogenesis.

cell biology↗

bioRxiv2021

Kinesin-4 Motor Teams Effectively Navigate Dendritic Microtubule Arrays Through Track Switching and Regulation of Microtubule Dynamics

Microtubules establish the directionality of intracellular transport by kinesins and dynein through their polarized assembly, but it remains unclear how directed transport occurs along microtubules organized with mixed polarity. We investigated the ability of the plus-end directed kinesin-4 motor KIF21B to navigate mixed polarity microtubules in mammalian dendrites. Reconstitution assays with recombinant KIF21B and engineered microtubule bundles or extracted neuronal cytoskeletons indicate that nucleotide- independent microtubule binding regions of KIF21B modulate microtubule dynamics and promote directional switching on antiparallel microtubules. Optogenetic recruitment of KIF21B to organelles in live neurons resulted in unidirectional transport in axons but bi-directional transport with a net retrograde bias in dendrites; microtubule dynamics and the secondary microtubule binding regions are required for this net directional bias. We propose a model in which cargo-bound KIF21B motors coordinate nucleotide- sensitive and insensitive microtubule binding sites to achieve net retrograde movement along the dynamic mixed polarity microtubule arrays of dendrites.

cell biology↗

bioRxiv2020

Cega: A Single Particle Segmentation Algorithm to Identify Moving Particles in a Noisy System

Improvements to particle tracking algorithms are required to effectively analyze the motility of biological molecules in complex or noisy systems. A typical single particle tracking (SPT) algorithm detects particle coordinates for trajectory assembly. However, particle detection filters fail for datasets with low signal-to-noise levels. When tracking molecular motors in complex systems, standard techniques often fail to separate the fluorescent signatures of moving particles from background noise. We developed an approach to analyze the motility of kinesin motor proteins moving along the microtubule cytoskeleton of extracted neurons using the Kullback-Leibler (KL) divergence to identify regions where there are significant differences between models of moving particles and background signal. We tested our software on both simulated and experimental data and found a noticeable improvement in SPT capability and a higher identification rate of motors as compared to current methods. This algorithm, called Cega, for find the object, produces data amenable to conventional blob detection techniques that can then be used to obtain coordinates for downstream SPT processing. We anticipate that this algorithm will be useful for those interested in tracking moving particles in complex in vitro or in vivo environments.

cell biology↗

bioRxiv2020

Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway

Autophagy is a degradative pathway required to maintain neuronal homeostasis. Neuronal autophagosomes form constitutively at the axon terminal and mature via lysosomal fusion during dynein-mediated transport to the soma. How the dynein-autophagosome interaction is regulated during maturation is unknown. Here, we identify a series of handoffs between dynein effectors as autophagosomes transit along the axons of primary neurons. In the distal axon, JIP1 initiates autophagosomal transport, while autophagosomes in the mid-axon require HAP1 and Huntingtin for motility. We demonstrate that HAP1 is a bonafide dynein activator, binding the dynein-dynactin complex via canonical and noncanonical interactions. JIP3 is found on most axonal autophagosomes but specifically regulates the transport of acidified autolysosomes. Inhibiting autophagosomal transport disrupts maturation, while inhibiting autophagosomal maturation perturbs the association and function of dynein effectors. Thus maturation and transport are tightly linked. These results describe a novel maturation-based dynein effector handoff on neuronal autophagosomes that is key to autophagosomal motility, cargo degradation, and the maintenance of axonal health. SummaryNeuronal autophagosomes form in the distal axon and mature via fusion with lysosomes during their dynein-driven transport to the soma. Dynein is regulated on autophagosomes by distinct effector proteins--JIP1, HAP1, and JIP3--depending on location and autophagosomal maturity. In this sequential pathway, transport and maturation state are tightly linked to maintain neuronal health.

cell biology↗